Compounds which modulate the Hedgehog pathway, which is the preferred pathway for cell differentiation, have the advantage of being able to be used to promote the differentiation of stem cells of embryonic or adult origin or multipotent cells. The repair of tissues damaged subsequent to a disease, a trauma or age increasingly uses stem cells or progenitor cells which retain the ability to differentiate into various cell types. These cells constitute a reservoir capable of renewing tissues in order to restore biological functions. Mesenchymal stem cells, for example, can give osteoblasts, chondrocytes, adipocytes or hematopoiesis-supporting stromal cells.
The techniques for directing these cells toward a chosen phenotype are generally laborious (transformation of cells using expression vectors and need to express several genes) and alternative solutions such as the use of small synthetic molecules which induce differentiation would constitute a promising track.
The Hedgehog (Hh) signaling molecule is a secreted protein which activates a signaling pathway which plays a fundamental role in the morphogenesis of numerous tissues and the development of the brain, and also in cell proliferation, and appears to be involved in tissue maintenance and repair in adults (Ingham and McMahon 2001; Wechlser-Reya and Scott 2001; Marti and Bovolenta 2002; Lum and Beachy 2004; Varjosalo and Taipale 2008).
Hh proteins are synthesized in the form of immature precursors of approximately 45 kDa which are subjected to an intramolecular cleavage catalyzed by the C-terminal region of the precursor. This cleavage produces a C-terminal fragment of 25 kDa without a known additional function and an active amino-terminal fragment of 19 kDa which has a cholesterol molecule bonded to its C-terminal end. This N-terminal fragment has all the known signaling activities of Hh proteins.
The Hh protein signaling pathway comprises three main components: the Hh ligand, a transmembrane receptor circuit, composed of the Patched negative regulator (Ptc) and the Smoothened activator (subsequently denoted without distinction Smo or Smoothened), and a cytoplasmic complex which regulates the transcriptional effectors.
In mammals, there are two Ptc genes encoding, respectively, Ptc1 and Ptc2, which are glycoproteins comprising 12 transmembrane domains, homologous to bacterial transporters. The product of the Smo gene which encodes a protein of the G protein-coupled receptor family has no known endogenous ligand. The exact mechanism of regulation of the Hedgehog pathway has not yet been completely elucidated. In the absence of Hh, Ptc appears to block the constitutive activity of Smo. The binding of Hh to Ptc appears to lift this inhibition and enable the transduction of the signal by means of Smo. The mechanism of regulation of the activity of Smo by Ptc could involve a molecule which is transported by Ptc and which interacts with Smo (Taipale et al. 2002). Activation of Gli transcription factors is involved in the cascade of events resulting from the activity of Smo. The type I transmembrane protein HIP (Hedgehog Interacting Protein) constitutes another receptor of Hh molecules and constitutes a negative regulator of the pathway (Ho and Scott 2002). Other genes, such as dispatched (DispA), in particular, appear to be involved in the release and accumulation in the extracellular medium of Hh proteins in soluble form (Ma et al. 2002). More recently, other membrane proteins such as Cdo and Boc have been described as capable of binding to Shh and of potentiating its effect (Ma et al. 2008).
The Hh protein and the associated transduction pathway, initially demonstrated in drosophila, are conserved in vertebrates and invertebrates. A single Hh homolog is present in drosophila, whereas three Hh homologs: Sonic (Shh), Indian (Ihh) and Desert (Dhh), are present in mammals. Among these three homologs, Shh has been most commonly studied owing to its widespread expression profile during development. Shh participates in ventralization of the neural tube by specifying the early phenotype of several neuronal types along the ventral median line (spinal cord motoneurons, dopaminergic or cholinergic neurons) and by inducing the generation of oligodendrocyte precursors from the ventral spinal cord (McMahon et al. 2003). Moreover, Shh induces the survival of gabaergic and dopaminergic neurons, directs the fate of serotininergic precursors and prevents dopaminergic neuron deaths caused by the MPP toxin. Finally, it induces the proliferation of granular cell precursors in the early post-natal cerebellum. The other members of the Hh family, for their part, participate respectively in the development of bone tissue (Ihh), of the testicles of peripheral nerves (Dhh). In addition, the results obtained with Shh also apply to Dhh and Ihh.
The regulatory role of the Hedgehog protein signaling pathway during embryonic development has been widely studied: Hh has been associated with the processes for maintaining and repairing normal tissue, and with the spatial temporal regulation of proliferation and differentiation, thus allowing developing tissues to reach their correct sizes with the appropriate cell types and appropriate degrees of vascularization and innervation. The essential role of the function of Hh is demonstrated by the dramatic consequences of defects in this signaling pathway in the human fetus, such as holoprosencephaly observed with Shh mutants (Traiffort et al. 2004).
The Shh pathway has been identified in the spinal cord and the adult brain where the amino-terminal active form of the molecule is expressed in numerous regions, at a level higher than that encountered during the early post-natal period (Traiffort et al. 1999; Traiffort et al. 2001; Coulombe et al. 2004).
Although the roles of Shh in adults have not been completely elucidated, its involvement in the maintenance and renewal of stem cells is increasingly studied (Charytoniuk et al. 2002; Ahn and Joyner 2005; Stecca and Ruiz i Altaba 2009). These stem cells have been identified in several regions of the adult brain, including the subvectricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampus. They provide new precursors throughout life, which have the ability to migrate respectively to the olfactory bulb and the granular cell layer. Under defined pathological conditions, these cells can deviate from their path to the damaged zone. Furthermore, Shh has a chemoattractive activity on neural precursors of the SVZ, an effect which is blocked by an Smo inhibitor (Argot et al. 2008). Shh also stimulates the proliferation of neural progenitors in the SGZ (Lai et al. 2003), but the results remain controversial for those of the SVZ. Moreover, Shh injected into the lateral ventricle of mice also makes it possible to increase the number of oligodendroglial precursors in the cortex, suggesting that activation of the pathway could be of therapeutic interest in demyelinating diseases (Loulier et al. 2006).
Finally, under pathological conditions, such as those observed in a model of Parkinson's disease or a model of peripheral neuropathy, Shh is capable of preserving the axonal projections of dopaminergic neurons in the striatum or of reducing the time necessary for motor recovery subsequent to crushing of the sciatic nerve (Pepinsky et al. 2002; Tsuboi and Shults 2002).
Dysfunctions of the Shh signaling pathway have been associated with many cancers, more particularly with basal cell carcinomas in the skin and with medulloblastomas in the brain. These tumors are most commonly related to mutations of various participants of the Hedgehog pathway, causing an overactivation thereof (Beachy et al. 2004; Scales and de Sauvage 2009). More generally, the location of these tumors is closely correlated with the sites of expression of the components of the pathway during embryonic development. By way of nonlimiting example, mention may be made of: breast cancers, meningiomas, glioblastomas, gastrointestinal cancers (in particular of the stomach), prostate cancers, ovarian fibromas and dermoids, rhabdomyosarcomas, small cell lung cancers, and oral squameous cell carcinomas. Recently, Shh has been associated with psoriasis.
Owing to the crucial role of the Hh protein signaling pathway in many physiological processes and, consequently, the significance of the pathological conditions associated with the dysfunction thereof, the components of this pathway represent targets for the development of new molecules capable of modulating, i.e. of activating or inhibiting, this pathway and therefore of positively or negatively regulating development, including the proliferation, the differentiation, the migration and the survival (apoptosis) and/or the activity of differentiated cells and of stem cells, in vitro and/or in vivo in the embryo or in adults.
Such molecules, in particular inhibiting molecules, are of use in the treatment of tumors associated with hyperactivation of the Hedgehog pathway: nervous tissue tumors (medulloblastomas, primitive neuroectodermal tumors, glioblastomas, meningiomas and oligodendrogliomas), skin tumors (basal cell carcinomas, trichoepitheliomas), muscle and bone tissue tumors (rhabdomyosarcomas, osteosarcomas) and tumors of other tissues (kidney, bladder).
Such molecules, in particular stimulating molecules, are also of use in the treatment of pathological conditions of neurodegenerative type involving the Hh pathway (Parkinson's disease, Huntington's chorea, Alzheimer's disease, multiple sclerosis, motoneuron disease), and diseases in which modulation of the Hh signaling pathway could be beneficial, such as diabetes.
Such molecules are also of use in the medical or surgical treatment (plastic or reconstructive surgery, tissue or organ transplant) of numerous acute, subacute or chronic, genetic or acquired pathological conditions—involving a tissue dysfunction related to a dysregulation of the Hh pathway-, for inducing the formation, regeneration, repair and/or increase in the activity of tissues such as, in a nonlimiting manner: the nervous tissue [central nervous system (brain) and peripheral nervous system (sensory, motor, sympathic neurons)], bone, cartilage, testicles, liver, spleen, intestine, pancreas, kidneys, smooth and squelletial muscles, heart, lungs, skin and body hair, mucus membranes, blood cells and immune system cells. By way of nonlimiting example of these pathological conditions, mention may in particular be made of neuropathies and associated neuromuscular diseases, diabetes, alopecia, burns, ulcerations (skin and mucosal) and spermatogenesis disorders.
Various molecules, capable of modulating the activity of the Hedgehog pathway, have been identified (the structures of some of these molecules will be represented hereinafter):                the Hh proteins and derived polypeptides which stimulate the pathway by acting on the Ptc protein;        agonists derived from oxysterols (Corcoran and Scott 2006) and smaller organic molecules such as SAG (Chen et al. 2002); the Hh molecules Ag1.2 (Frank-Kamenetsky et al. 2002) or else purmorphamine (Sinha and Chen 2006), the activity of which on the Smo receptor has been established. Recently, more active Ag1.2 derivatives have been described (Brunton et al. 2009);        nitrogenous or non-nitrogenous heterocyclic organic molecules which inhibit the Hedgehog pathway (see international applications WO 2007/054623, WO 2007/059157, WO 01/74344, WO 01/19800, WO 01/26644, WO 02/30421, WO 2005/033288, WO 2005/042700, WO 2008/014291 and WO 2007/120827); a compound of this type is currently in clinical testing (GDC-0449 described from WO 2006/028958);        benzamidazole derivatives (WO 2008/075196);        plant steroids extracted from Veratrum spp (cyclopamine, jervine, etc.) and derivatives thereof which inhibit the pathway (see the international patents and applications U.S. Pat. No. 6,432,970, WO 99/52534, WO 01/27135, U.S. Pat. No. 6,291,516, WO 00/41545 and WO 02/30462 and Taipale et al., 2000 and Berman et al. 2002). However, cyclopamine is a teratogenic agent responsible for holoprosencephaly and cyclopia in the embryo in mammals, and an absence of toxicity, for mammals, of the other plant steroid-derived compounds has not yet been demonstrated;        mifepristone (17β-hydroxy-11β-(4-dimethylamino-phenyl)-17α-(prop-1-ynyl)estra-4,9-dien-3-one), also known as RU-486 or RU-38486 (see application FR 03/00646) for which an inhibitory activity on the activity of the Hh protein signaling pathway has been demonstrated;        N-acylthiourea and N-acylurea molecules which inhibit the Hedgehog protein signaling pathway (see application FR 08/02302).        

Structure of Compounds which are Modulators of the Hedgehog pathway:
The SAG compound is an agonist of the pathway, whereas the compounds cyclopamine, Cur61414, GDC-0449, SANT-1, MRT-14, MRT-79 and MRT-81 are antagonists; all these compounds bind to the Smo receptor.
The hedgehog-pathway-modulating molecules act predominantly on the Smoothened protein, as it has been possible to show by means of binding experiments, optionally in competition with a known compound; numerous recent publications have provided evidence of conformational modifications of the Smo receptor and some of these modulators appear to act preferentially on one or another of these conformations.
The transmission of the Hh signal by the Smo receptor and the activation of the target genes appear to be associated with trafficking of this receptor to the primary cilium. This trafficking can be stimulated by the action of Hh itself, but also of small stimulatory molecules such as SAG (Rohatgi et al. 2007) or even small inhibitory molecules such as cyclopamine. Conversely, other inhibitors prevent the translocation of the receptor to the cilium (Rohatgi et al. 2009; Wang et al. 2009; Wilson et al. 2009). The latter authors have suggested that the receptor expressed at the cilium originates essentially from intracellular sources via an intraflagellar transport (IFT transport) system (Wang et al. 2009).
These data using small molecules have led Rohatgi et al. to propose a two-step model: a step of translocation to the cilium and then a second step of activation of the receptor. These two steps can be pharmacologically differentiated since various antagonists can differentially block each step (Rohatgi et al. 2009). Similarly, Wilson et al. suggest that Smo exists in several active or inactive conformations which influence its location and its transport to the cilium (Wilson et al. 2009).
From the molecular point of view, Zhao et al. have shown that the inactive form of Smo is maintained by molecular electrostatic interactions maintained by arginine clusters in the cytoplasmic tail. Following the action of Hh, the receptor is phosphorylated and these interactions are disrupted. The conformational change then occurs and induces dimerization of the cytoplasmic tails required for the activation of Smo (Zhao et al, 2007).
These authors suggest that, depending on the number of arginine clusters inhibited by differential phosphorylation, the Smo receptor could operate as a molecular rheostat of Hh signaling.
The conformation of the Smo receptor appears to be particularly linked to the action of pharmacological compounds. It has recently been shown that two known antagonists of the receptor: SANT-1 and SANT-2, act allosterically on distinct sites of the receptor (Rominger et al. 2009). Moreover, it has been published that simply changing a methyl to a propyl or allyl on the SAG agonist makes it possible to convert this molecule into powerful antagonists (IC50=20-70 nM, SANT75, SANT74), by regulating the conformation of the Smo receptor (Yang et al. 2009).